RNA-dependent RNA polymerases (RdRP) play critical roles in RNA-mediated gene silencing. In Arabidopsis thaliana, RNA-Dependent RNA Polymerase 2 (RDR2) generates double-stranded RNAs (dsRNAs) from a single stranded RNA template generated by DNA-dependent nuclear RNA Polymerase IV (Pol IV). Resulting dsRNAs are then processed into short-interfering RNAs that guide RNA-directed DNA methylation and transcriptional gene silencing. We determined the structure of RDR2 at 3.1 Å resolution by single-particle cryo-electron microscopy. The structure suggests its implications for double-stranded RNA synthesis in gene silencing. The seminar will also touch on several structure examples determined by Takagi lab using cryo-EM including Mediator CDK8 module from the yeast Saccharomyces cerevisiae, human asparagine synthetase, and multicopper oxidase from Marinithermus hydrothermalis.
“Focused Ion Beam Scanning Electron Microscopes for Volume-EM and Cryo-Microscopy”
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Date:
3:30 PM – 5:00 PM Tuesday, September 19th, 2023 (JST),
Location: Hybrid (CryoEM-Lab KEK and Zoom)
Speaker
Dr. Alexander Rigort
Thermo Fisher Scientific, Munich, Germany
Abstract
FIB/SEM microscopes have proven to be indispensable tools for cryo-electron microscopy and volumetric imaging of biological specimens. These devices can be used to analyze samples at room temperature and under cryo-conditions. Equipped with automation software for both data acquisition and electron transparent lamella fabrication, FIB/SEM microscopes offer a wide range of applications and multiple workflows for obtaining volumetric information. The imaging capabilities include high-resolution serial imaging with FIB/SEM tomography (slice & view), millimeter-scale analysis with the spin mill method, array tomography and correlative microscopy with the iFLM system. Additionally, Plasma-FIB systems allow the use of up to four different ion species, which can be used to optimize milling and imaging for both cryogenic and resin-embedded samples. In this lecture, I will provide an overview of the current state of developments, applications, and the related FIB/SEM product range.
Profile:
Dr. Alex Rigort works in product marketing and development for Thermo Fisher Scientific and holds a guest researcher position at the Max Planck Institute of Biochemistry in Martinsried. He has pioneered the use of cryo-focused ion beam instruments for cryo-tomography at the Max Planck Institute of Biochemistry and has long-standing experience in cryo-electron microscopy.
本PF研究会では、「KEKにおけるクライオ電子顕微鏡：これまでの5年間とこれから（Cryo-EM in KEK: 5-year history and future）」と題し、KEKにおけるクライオ電子顕微鏡に関するこれまでの取り組みを紹介するとともに、放射光実験との連携などを含めて今後の展開についてユーザーの皆様と共に議論を深めたいと考えています。また、本PF研究会に合わせて、KEKのクライオ電顕ユーザーとの意見交換のために、KEKクライオ電顕ユーザーミーティングを開催したいと考えています。
“Ambient-Temperature Time-Resolved Serial Femtosecond Crystallography Studies of Bacterial Ribosome Complexes”
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Date:
3:30 PM – 5:00 PM Thursday, July 27th, 2023 (JST),
Location: Hybrid (CryoEM-Lab KEK and Zoom)
Speaker
Hasan Demirci
Hasan DeMirci, PhD
Assistant Professor, Koc University, Istanbul, Turkey.
Affiliate, Stanford PULSE Institute, SLAC National Accelerator Laboratory, CA, USA.
Abstract
High-resolution ribosome structures determined by cryo X-ray crystallography have provided important insights into the mechanism of translation. Such studies have thus far relied on large ribosome crystals kept at cryogenic temperatures to reduce radiation damage. We use serial femtosecond X-ray crystallography (SFX) with an X-ray free-electron laser (XFEL) to obtain diffraction data from ribosome microcrystals in liquid suspension at ambient temperature. Small 30S ribosomal subunit microcrystals programmed with initiation and decoding complexes and bound to either antibiotic compounds or their next-generation derivatives diffracted to high resolution. Our results demonstrate the feasibility of using SFX to better understand the structural mechanisms underpinning the interactions between ribosomes and other substrates such as antibiotics, initiation and decoding complexes. We have determined the structure of a large (50S) ribosomal subunit in a record-short time by using the record-low amount of sample during an XFEL beamtime. This structure is the largest one solved to date by any FEL source to near-atomic resolution (3 MDa). We expect that these results will enable routine structural studies, at near-physiological temperatures, of the large ribosomal subunit bound to clinically relevant classes of antibiotics targeting it, e.g. macrolides and ketolides, also with the goal of aiding the development of the next generation of these classes of antibiotics. Overall, the ability to collect diffraction data at near-physiological temperatures promises to provide new fundamental insights into the structural dynamics of the ribosome and other medically important drug targets with their functional and inhibitor complexes.
Profile:
2008. Ph.D., Molecular Biology, Cell Biology and Biochemistry, Brown University, RI, USA
島野先生は、脂質合成転写因子であるSterolregulatory-element binding proteins（SREBPs）の研究を続けてこられていますが（Shimano H. Nat Rev Endocrinol, 2017）その新規標的遺伝子の探索過程において、炭素数12-16 の飽和・一価不飽和脂肪酸を基質とする脂肪酸伸長酵素Elovl6 のクローニングに成功されています（Matsuzaka T et al. J Lipid Res, 2002）。この欠損マウスでは肝臓の脂肪酸組成が変化しますが 食餌性および遺伝性肥満によるインスリン抵抗性が、肥満が持続した状態でも改善することが明らかになりました（Matsuzaka T et al. Nat Med, 2007）。これをきっかけに、臓器脂肪酸の鎖長が、種々の臓器における様々な疾患に与える影響から新しい疾患治療理念を展開されています。
“Structural insights into the peroxisomal import machinery”
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Date:
4:00 PM – 5:30 PM Friday, April 7th, 2023 (JST),
Location: CryoEM-Lab KEK and Zoom
Speaker
Prof. Christos Gatsogiannis, PhD
Institute for Medical Physics and Biophysics and
Center for Soft Nanoscience (SoN),
University of Münster, Germany.
Abstract
Our research focuses on the molecular understanding of peroxisomal biogenesis. Peroxisomes are dynamic small organelles found in almost all eukaryotes and are capable of performing a plethora of crucial metabolic functions. Peroxisomal enzymes are synthesized on free cytoplasmic ribosomes and later imported into the peroxisomal lumen by the peroxisomal import machinery. However, the main characteristics of the peroxisomal import process remain poorly understood. Our research aims to characterize and visualize these processes using cryo-electron microscopy (single particle analysis and tomography) and biochemical and biophysical methods to understand, in order to provide the necessary mechanistic insights into this central mechanism. In this lecture, Prof. Gatsogiannis will talk about the current structural work of his group on the peroxisomal import machinery.
Profile:
2005 Master (Biology), University of Mainz, Germany
2009 Ph.D. (Biology), University of Mainz, Germany, 2009
2010 Postdoctoral Researcher with Prof. J. Markl, University of Mainz, Germany
2010-2015 Postdoctoral Researcher with Dr. S. Raunser,
Max Planck Institute for Molecular Physiology, Germany
2016-2020 Tenured Project Group Leader,
Max Planck Institute of Molecular Physiology, Germany
2020- Full Professor, University of Münster, Germany
2023- Co-Director, Center for Soft Nanoscience, Münster, Germany
